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1.
Anal Biochem ; 472: 45-51, 2015 Mar 01.
Artículo en Inglés | MEDLINE | ID: mdl-25461479

RESUMEN

Glucagon-like peptide-1 (GLP-1) is an incretin peptide that regulates islet hormone secretion. During recent years, incretin-based therapies have been widely used for patients with type 2 diabetes. GLP-1 peptides undergo N- and C-terminal processing for gain or loss of functions. We developed a method to quantify picomolar quantities of intact GLP-1 peptides using liquid chromatography-tandem mass spectrometry (LC-MS/MS). By employing this label-free selected reaction monitoring (SRM) method, we were able to analyze secreted GLP-1(1-37), GLP-1(7-37), and GLP-1(7-36 amid from human enteroendocrine NCI-H716 cells after stimulation with nateglinide, glucose, and sucralose. The absolute total concentrations of secreted GLP-1 peptides at baseline and after stimulation with nateglinide, glucose, and sucralose were 167.3, 498.9, 238.3, and 143.1 pM, respectively. Meanwhile, the ratios of GLP-1(1-37), GLP-1(7-37), and GLP-1(7-36 amide) to total GLP-1 peptides were similar (6 ± 3, 26 ± 3, and 78 ± 5%, respectively). The SRM assay can analyze the concentrations of individual GLP-1 peptides and, therefore, is a tool to investigate the physiological roles of GLP-1 peptides. Furthermore, the molecular species secreted from NCI-H716 cells were unknown. Therefore, we performed a secretopeptidome analysis of supernatants collected from cultured NCI-H716 cells. Together with GLP-1 peptides, we detected neuroendocrine convertase 1, which regulates peptide hormones released from intestinal endocrine L-cells.


Asunto(s)
Péptido 1 Similar al Glucagón/análisis , Péptido 1 Similar al Glucagón/metabolismo , Espectrometría de Masas , Proteómica/métodos , Línea Celular , Cromatografía Liquida , Humanos
2.
Biomed Chromatogr ; 24(7): 683-91, 2010 Jul.
Artículo en Inglés | MEDLINE | ID: mdl-19830681

RESUMEN

An automated method for high-throughput amino acid analysis, using precolumn derivatization high-performance liquid chromatography/electrospray mass spectrometry (HPLC/ESI-MS), was developed and evaluated. The precolumn derivatization step was performed in the reaction port of a home-built auto-sampler system. Amino acids were derivatized with 3-aminopyridyl-N-hydroxysuccinimidyl carbamate, and a 3 microm Wakosil-II 3C8-100HG column (100 x 2.1 mm i.d.) was used for separation. To achieve a 13 min cycle for each sample, the derivatization and separation steps were performed in parallel. The results of the method evaluation, including the linearity, and the intra- and inter-precision, were sufficient to measure physiological amino acids in human plasma samples. The relative standard deviations of typical amino acids in actual human plasma samples were below 10%.


Asunto(s)
Aminoácidos/análisis , Cromatografía Líquida de Alta Presión/métodos , Espectrometría de Masa por Ionización de Electrospray/métodos , Aminoácidos/sangre , Aminoácidos/metabolismo , Cromatografía Líquida de Alta Presión/instrumentación , Humanos , Espectrometría de Masa por Ionización de Electrospray/instrumentación
3.
Mol Immunol ; 38(10): 747-57, 2002 Feb.
Artículo en Inglés | MEDLINE | ID: mdl-11841834

RESUMEN

We have been proposing the functional distinction of two classes of macrophages (Mp), namely the reductive macrophages (RMp) with high intracellular content of glutathione (GSH) and the oxidative macrophages (OMp) with reduced content. At the same time we have been investigating the variation of RMp/OMp balance during aging of mice, especially in relation to the age related onset of autoimmune diseases. In this paper we have investigated the Th1/Th2 balance of thioredoxin (TRX) transgenic (Tg) mice, with prolonged life longevity, during aging in the context of the intracellular redox status of Mp, which has been hypothesized to be crucial in regulating the Th1/Th2 balance. It was confirmed that peritoneal resident Mp of Tg mice showed the higher GSH/GSSG ratios compared with that of age matched wild type (WT) mice. The predominance of RMp was associated with the sustained maintenance of Th1 prevalence during aging until 2 years in Tg mice, whereas WT littermates showed rapid polarization to Th2 around the age of 8 months. The Tg mice showed elevation of IFN-gamma and reduction of IL-10 with moderate change of IL-4 produced by CD4+ T cells. The WT mice showed inverse changes of IFN-gamma/IL-4 and IFN-gamma/IL-10 ratios during aging. In addition, IL-10 production by Mp was dramatically reduced in aged Tg mice. Thus, TRX Tg mice may be useful to investigate the contribution of the anti-oxidant defense mechanism during aging accompanied with increasing oxidative stress.


Asunto(s)
Envejecimiento/inmunología , Envejecimiento/metabolismo , Macrófagos Peritoneales/fisiología , Células TH1/inmunología , Células Th2/inmunología , Tiorredoxinas/genética , Animales , Regulación de la Expresión Génica/inmunología , Interferón gamma/inmunología , Interferón gamma/metabolismo , Interleucina-10/inmunología , Interleucina-10/metabolismo , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Ratones Transgénicos , Oxidación-Reducción , Compuestos de Sulfhidrilo , Células TH1/metabolismo , Células Th2/metabolismo , Tiorredoxinas/inmunología , Tiorredoxinas/metabolismo
4.
J Nutr ; 136(6 Suppl): 1716S-1721S, 2006 06.
Artículo en Inglés | MEDLINE | ID: mdl-16702345

RESUMEN

Although many animal studies have reported that dietary excess of methionine causes toxic changes including growth suppression and hemolytic anemia, the biochemical mechanism and biomarkers for methionine toxicity have not been well elucidated. The present study aimed to identify toxicity biomarkers from plasma metabolites in rats fed excessive methionine. Young growing rats were fed graded doses of additional methionine for 2 wk. Cluster analysis of multivariate correlations was performed on the physiological and toxicity variables with plasma metabolites detected by GC/MS, amino acid analyzer, and thiol-specific analysis. Indicative variables for hemolysis such as splenic nonheme iron content and plasma bilirubin were grouped in the same cluster as many methionine metabolites. Homocysteine and some undefined metabolites in this cluster were found to be strong discriminators between nontoxic and toxic levels of methionine intake. Product-to-precursor ratios of each methionine metabolite demonstrated that excessive methionine intake caused a marked decrease only in the ratio of cystathionine to homocysteine, suggesting that metabolism from homocysteine to cystathionine would be rate limiting in the disposal of excessive methionine. Collectively from these results, homocysteine appeared to be the most plausible biomarker to assess methionine excess as a surrogate marker both for toxicity and for setting a metabolic upper limit.


Asunto(s)
Biomarcadores/sangre , Metionina/toxicidad , Aminoácidos/sangre , Análisis de Varianza , Animales , Bilirrubina/sangre , Cistationina/sangre , Dieta , Cromatografía de Gases y Espectrometría de Masas , Glicina/administración & dosificación , Hemólisis , Homocisteína/sangre , Hierro/sangre , Hierro/metabolismo , Masculino , Metionina/administración & dosificación , Ratas , Ratas Endogámicas F344 , Serina/administración & dosificación , Bazo/metabolismo
5.
J Nutr ; 135(6 Suppl): 1571S-5S, 2005 06.
Artículo en Inglés | MEDLINE | ID: mdl-15930471

RESUMEN

Changes were investigated in plasma metabolites and physiological and toxicological variables in rats fed for 2 wk on a basal diet or diets with 1.5, 5, 10, 15, and 30% added leucine. In the same experiment, the changes in gene expression in livers of rats fed the basal diet or diets with 5% and 15% added leucine were investigated using DNA microarrays. Cluster analysis of multivariate correlations of metabolites and physiological and toxicological variables indicated that the variables associated with excess nitrogen clustered together with leucine and alpha-ketoisocaproate. The gene expression data, although preliminary, indicated that there was little change in the expression of enzymes of the catabolic pathways for leucine but that there were changes in enzymes associated with nitrogen metabolism and other pathways downstream of leucine catabolism. The data seem consistent with excess leucine exerting its effects through the overloading of nitrogen metabolism and that urea or alpha-ketoisocaproate could be an early marker for the upper limit of adequate intake.


Asunto(s)
Aminoácidos/sangre , Perfilación de la Expresión Génica , Leucina/toxicidad , Hígado/efectos de los fármacos , Análisis de Secuencia por Matrices de Oligonucleótidos , Aminoácidos/orina , Animales , Dieta , Relación Dosis-Respuesta a Droga , Cromatografía de Gases y Espectrometría de Masas , Glucosa/metabolismo , Cetoácidos/metabolismo , Leucina/administración & dosificación , Leucina/metabolismo , Hígado/metabolismo , Masculino , Ratas , Ratas Endogámicas F344
6.
Eur J Immunol ; 33(4): 1001-11, 2003 Apr.
Artículo en Inglés | MEDLINE | ID: mdl-12672066

RESUMEN

The redox status of macrophages (M phi), indexed by intracellular content of glutathione (icGSH), varies sequentially along with disease progression in nonobese diabetic mice. At the stage of early insulitis, M phi skew to oxidative M phi (OM phi) with decreased icGSH, then to reductive M phi (RM phi) with elevated icGSH and to OM phi after the occurrence of diabetes. RM phi or OM phi inducing agents either delayed or accelerated the onset of diabetes in a mutually inverse manner. RM phi or OM phi adoptively transferred exacerbated or ameliorated the disease progression depending on the redox status of M phi of recipient mice. The new paradigm that the sequential conversion of redox status of M phi dictates the pathological progression may provide a new insight on the mechanism underlying autoimmune diabetes.


Asunto(s)
Diabetes Mellitus Tipo 1/inmunología , Macrófagos/metabolismo , Traslado Adoptivo , Animales , Ciclofosfamida/farmacología , Diabetes Mellitus Tipo 1/metabolismo , Diabetes Mellitus Tipo 1/patología , Progresión de la Enfermedad , Femenino , Glutatión/análisis , Cinética , Macrófagos/química , Macrófagos/trasplante , Ratones , Ratones Endogámicos NOD , Oxidación-Reducción/efectos de los fármacos , Fenotipo , Compuestos de Sulfhidrilo/farmacología , Células TH1/inmunología , Células Th2/inmunología
7.
J Nutr ; 134(6 Suppl): 1651S-1655S; discussion 1664S-1666S, 1667S-1672S, 2004 06.
Artículo en Inglés | MEDLINE | ID: mdl-15173446

RESUMEN

We report on research progress on two approaches that may be useful in determining the upper adequacy range for macronutrients such as amino acids. One approach was to attempt to identify "toxic metabolites" that were responsible for toxicity or biomarkers for the toxicity of excessive intake of an amino acid in rats. We found that there was hepatic toxicity that was specifically associated with L-cystine excess, but not with L-cysteine excess. We analyzed urine samples from rats fed basal diets or L-cystine or L-cysteine excess diets and identified 25 peaks from gas chromatography mass spectrometry analysis that were specific for L-cystine excess and also correlated with toxicity markers. Another approach was to try to identify "metabolic limits" by measuring CO(2) arising from amino acid excess. Uniformly (13)C labeled L-leucine was used as tracer, in diets with added L-leucine fed to rats, and (13)CO(2) arising from its metabolism was collected over 24 h and the fraction of the ingested L-leucine that was exhaled as CO(2) was calculated. The fractional exhalation of (13)CO(2) increased with increasing L-leucine dose, but showed an inflexion point at approximately 8.9 g/kg body weight, after which it reached a plateau. This suggested that >8.9 g/kg BW, the catabolism of L-leucine changed and this approximately coincided with the dose above which a statistically significant decrease in body weight was seen.


Asunto(s)
Aminoácidos/administración & dosificación , Fenómenos Fisiológicos Nutricionales de los Animales , Evaluación Nutricional , Estado Nutricional , Animales , Masculino , Necesidades Nutricionales , Ratas , Ratas Endogámicas F344
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